Hiroshi Suehisa

Presence of Epidermal Growth Factor Receptor Gene T790M Mutation as a Minor Clone in Non–Small Cell Lung Cancer

The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only 1 T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 x 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of 111 cases) than in early-stage tumors (1 of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.

The impact of sex and smoking status on the mutational spectrum of epidermal growth factor receptor gene in non-small cell lung cancer

Purpose: Mutation of epidermal growth factor receptor (EGFR) gene has been reported to be present in non–small cell lung cancer (NSCLC) and significantly associated with female sex and never-smoking status. In this study, we extensively investigated the impact of sex and smoking on the EGFR mutation. Experimental Design: We examined EGFR exons 18 to 21 status in 1,467 NSCLC patients by direct sequencing to study the impact of sex and smoking status on the EGFR mutational spectrum. Results: Among 1,467 patients, 197 mutations were found at exon 19, 176 at exon 21, 21 at exon 18, and 24 at exon 20. To examine the independent effect of sex and smoking, the mutational status of each exon was compared between smokers and never smokers in each sex and between males and females stratified by smoking status. In females, exon 19 (P = 0.001) and exon 21 (P < 0.001) mutations were significantly less frequent in ever smokers compared with never smokers. In males, exon 19 (P < 0.001), exon 21 (P < 0.001), and exon 18 (P = 0.003) mutations were significantly less frequent in ever smokers compared with never smokers. In analysis stratified by smoking, there was no difference in sex among never smokers. However, exon 19 mutations were significantly less frequent in males compared with females among ever smokers (P = 0.003). In addition, the interactive effect of male sex and ever smoking status significantly decreased the frequency of exon 19 mutations (P = 0.047) when female never smoker was set as a reference. Conclusion: Both sex and smoking status could influence the EGFR mutational spectrum. Our findings suggest that individual EGFR exons may have differing susceptibilities for mutagenesis.

Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung

Introduction: We investigated EGFR and KRAS alterations among atypical adenomatous hyperplasia and small lung adenocarcinomas with bronchioloalveolar features to understand their role during multistage pathogenesis. Methods: Sixty lesions measuring 2 cm or less were studied, including 38 noninvasive lesions (4 atypical adenomatous hyperplasias, 19 Noguchi type A and 15 type B) and 22 invasive lesions (type C) based on the World Health Organization classification and Noguchis criteria. EGFR and KRAS mutations were examined using PCR-based assays. EGFR copy number was evaluated using fluorescence in situ hybridization. Results: EGFR and KRAS mutations were found in 26 (43.3%) and 5 (8.3%) lesions, respectively. Increased EGFR copy number status was identified in 10 lesions (16.7%), both mutant and wild type. EGFR or KRAS mutations were present in 39.5% and 7.9% (respectively) of noninvasive lesions and 50% or 9.1% (respectively) of invasive lesions. EGFR copy number was increased in 7.9% and 31.8% of noninvasive and invasive lesions (P = 0.029). Multivariate analysis revealed that increased EGFR copy number was the only significant factor to associate with invasive lesions (P = 0.035). Conclusions: EGFR and KRAS mutations occur early during the multistage pathogenesis of peripheral lung adenocarcinomas. By contrast, increased EGFR copy number is a late event during tumor development and plays a role in the progression of lung adenocarcinoma independent of the initiating molecular events.

Risk Factors for Recurrence and Unfavorable Prognosis in Patients with Stage I Non-small Cell Lung Cancer and a Tumor Diameter of 20 mm or Less

Background: The purpose of this study was to identify risk factors for disease recurrence and unfavorable prognosis after surgical resection for stage I non-small cell lung cancer in patients with tumor diameters of ≤20 mm. Methods: One hundred sixty-three patients who had pathologic stage I non-small cell lung cancer with tumor diameters ≤20 mm and who had undergone a lobectomy with mediastinal lymph node dissection were retrospectively reviewed. The relationships between clinicopathologic factors and clinical outcomes, including recurrence and survival, were then examined. The clinicopathologic factors examined in this study were age, sex, smoking status, preoperative serum carcinoembryonic antigen level, pathologic tumor size, histologic subtype, histologic grade, and visceral pleural invasion. Results: Among the clinicopathologic factors that were examined, the histologic grade of the carcinoma status was significantly related to a high risk of recurrence when analyzed using univariate (p = 0.01) and multivariate analyses (p = 0.049). Regarding survival, patients with poorly differentiated carcinomas showed a significantly unfavorable overall survival (p < 0.001), disease-specific survival (p = 0.003), and disease-free survival (p = 0.002) compared with patients with well-/moderately differentiated carcinomas according to univariate analyses. A Cox proportional hazards model indicated that a poorly differentiated carcinoma status was the only independent factor for an unfavorable overall survival (p = 0.02), disease-specific survival (p = 0.046), and disease-free survival (p = 0.04). Conclusions: Poor differentiation of tumor was the only risk factor for recurrence and an unfavorable prognosis for stage I non-small cell lung cancer patients with tumor diameters of ≤20 mm.

Detection of EGFR gene mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients.

Introduction: In this study, we examined whether epidermal growth factor receptor (EGFR) mutations were detectable using a polymerase chain reaction-based assay and wash fluid of computed tomography (CT)-guided lung biopsy needles. Methods: DNA was extracted from wash fluid of CT-guided biopsy needles of 53 lung tumors (as diagnosed according to the results of the CT-guided biopsies). EGFR mutations, specifically exon19 deletions and exon21 L858R mutations, were examined using a mutant-enriched polymerase chain reaction assay. We also examined the presence of EGFR mutations in 26 surgically resected tumor specimens and compared the results with those obtained for the corresponding wash fluid samples. Results: The amount of DNA obtained for the wash fluid of the CT-guided biopsy needles ranged from 35 to 2360 ng. There were no significant differences in the amount of extracted DNA according to the tumor characteristics, including tumor size and the percentage of ground glass opacity. Thirty-four of the 53 lung tumor samples were histologically diagnosed as non-small cell lung cancer (NSCLC). Exon19 deletions and exon21 L858R mutations in EGFR were detected in 4 (12%) and 13 (38%) of 34 NSCLC cases, respectively. No EGFR mutations were found in the non-NSCLC cases. The EGFR mutation status in the wash fluid samples was consistent with those obtained for all 26 corresponding surgical specimens. Conclusion: Our results indicate that EGFR mutations can be detected using wash fluid of CT-guided biopsy needles. In this manner, the DNA genotype can be determined even in extremely small clinical specimens using highly sensitive assays.

Epidermal Growth Factor Receptor Mutation Status and Adjuvant Chemotherapy With Uracil-Tegafur for Adenocarcinoma of the Lung

PURPOSE Adjuvant chemotherapy with uracil-tegafur has been demonstrated to prolong survival among patients with resected lung adenocarcinomas. Epidermal growth factor receptor (EGFR) mutations have been reported to be present in lung adenocarcinomas. The present study evaluated whether the EGFR status could be used as a biologic predictor of the outcome of adjuvant chemotherapy with uracil-tegafur. PATIENTS AND METHODS The EGFR mutational status of 187 patients with resected lung adenocarcinomas was determined using a polymerase chain reaction-based assay for EGFR exons 19 and 21; the results were then correlated with the effect of adjuvant uracil-tegafur chemotherapy on survival. The antiproliferative effect of fluorouracil (FU) on adenocarcinoma cell lines with EGFR wild-type or mutant type status was examined by measuring the inhibitory concentrations at 50% (IC(50)s). RESULTS Among the 187 patients, 68 received uracil-tegafur as adjuvant chemotherapy, and 119 were not treated with any chemotherapeutic agents. EGFR mutations were present in 79 patients (43%). Overall, the adjuvant chemotherapy with uracil-tegafur significantly prolonged survival compared with the control group (hazard ratio = 0.38; P = .005). The survival benefit of adjuvant chemotherapy with uracil-tegafur was also examined after stratifying the patients according to EGFR mutation status. Adjuvant chemotherapy significantly prolonged survival among patients with EGFR wild-type tumors (hazard ratio = 0.34; P = .013) but not among patients with EGFR mutant tumors. In an in vitro experiment, the IC(50)s of EGFR mutant cells to FU were higher than those of wild-type cells, indicating that EGFR wild-type cells are more sensitive to FU than mutant cells. CONCLUSION EGFR status influenced the effect of adjuvant chemotherapy with uracil-tegafur. Adjuvant chemotherapy could be customized based on EGFR status.

Natural History of Pulmonary Subsolid Nodules: A Prospective Multicenter Study

Introduction: The purpose of this study was to evaluate the natural course of the progression of pulmonary subsolid nodules (SSNs). Materials and Methods: Eight facilities participated in this study. A total of 795 patients with 1229 SSNs were assessed for the frequency of invasive adenocarcinomas. SSNs were classified into three categories: pure ground‐glass nodules (PGGNs), heterogeneous GGNs (HGGNs) (solid component detected only in lung windows), and part‐solid nodules. Results: The mean prospective follow‐up period was 4.3 ± 2.5 years. SSNs were classified at baseline as follows: 1046 PGGNs, 81 HGGNs, and 102 part‐solid nodules. Among the 1046 PGGNs, 13 (1.2%) developed into HGGNs and 56 (5.4%) developed into part‐solid nodules. Among the 81 HGGNs, 16 (19.8%) developed into part‐solid nodules. Thus, the SSNs at the final follow‐up were classified as follows: 977 PGGNs, 78 HGGNs, and 174 part‐solid nodules. Of the 977 PGGNs, 35 were resected (nine minimally invasive adenocarcinomas [MIAs], 21 adenocarcinomas in situ [AIS], and five atypical adenomatous hyperplasias). Of the 78 HGGNs, seven were resected (five MIAs and two AIS). Of the 174 part‐solid nodules, 49 were resected (12 invasive adenocarcinomas, 26 MIAs, 10 AIS, and one adenomatous hyperplasia). For the PGGNs, the mean period until their development into part‐solid nodules was 3.8 ± 2.0 years, whereas the mean period for the HGGNs was 2.1 ± 2.3 years (p = 0.0004). Conclusion: This study revealed the frequencies and periods of development from PGGNs and HGGNs into part‐solid nodules. Invasive adenocarcinomas were diagnosed only among the part‐solid nodules, corresponding to 1% of all 1229 SSNs.

Impact of HER2 and EGFR gene status on gefitinib-treated patients with nonsmall-cell lung cancer

We previously examined the relationship between epidermal growth factor receptor (EGFR) status and clinical outcomes of nonsmall‐cell lung cancer (NSCLC) patients treated with gefitinib. In our study, we additionally examined HER2 status and investigate the impact of genetic status as predictors in Japanese NSCLC patients treated with gefitinib. The HER2 copy number status was determined by fluorescence in situ hybridization assay and mutation of HER2 exon 20 was determined by direct sequencing in 74 patients to investigate the relationship between molecular statuses including HER2 and EGFR and the clinical outcomes of patients treated with gefitinib. The high HER2 copy number was identified in 32 (43.2%) of 74 NSCLC patients and no HER2 exon 20 mutations were found. The high HER2 copy number was significantly associated with the sensitivity to gefitinib (p = 0.0045) and a prolonged progression‐free survival (PFS) (p = 0.0089) in a univariate analysis, but not in a multivariate analysis. Multivariate analyses exhibited that the drug‐sensitive EGFR mutation was an independent predictive factor of a better sensitivity to gefitinib (OR = 41.9, p = 0.002), prolonged overall survival (HR = 0.32, p = 0.019) and PFS (HR = 0.31, p = 0.0045). The high EGFR copy number might have a weak association with better response and prognosis without statistical significance. In conclusion, the drug‐sensitive EGFR mutation, rather than HER2 and EGFR copy numbers, is a determinant of favorable clinical outcomes in gefitinib‐treated patients with NSCLC, although the high HER2 copy number, to some extent, may influence the gefitinib effect.

Solitary fibrous tumor of the mediastinum

An 18-year-old man was referred to our hospital for further evaluation of a right anterior mediastinal tumor that measured 6 cm in diameter. Computed tomography-guided transcutaneous aspiration biopsy was performed, but no definitive diagnosis could be obtained. Because the tumor did not appear to be a high-grade malignant tumor, we undertook resection of the tumor to obtain a definitive diagnosis and provide appropriate treatment. Total thymectomy with tumor resection was performed through a median sternotomy. The tumor was solid, measuring 5.2 × 4.2 × 3.5 cm. The histological diagnosis was solitary fibrous tumor (SFT) arising from the mediastinum. Most extrathoracic SFTs appear to pursue a benign course, although careful long-term follow-up of these patients is necessary because the tumors have been reported to recur or metastasize in some cases.

Current status of postoperative follow-up for lung cancer in Japan: questionnaire survey by the Setouchi Lung Cancer Study Group-A0901.

PurposeThere is no recommended standard follow-up program after resection for lung cancer. Under these circumstances, each doctor establishes his or her own follow-up protocol. This questionnaire survey was conducted to grasp the current status of postoperative follow-up in Japan.MethodsThe questionnaire survey was aimed at determining what examinations were performed and at what frequencies in the setting of postoperative follow-up. Based on these results, examinations performed at a frequency of >50% and the time points after resection at which they were performed were selected and presented as components of an average follow-up program.ResultsQuestionnaires were sent to 44 institutions, and 26 doctors responded to the questionnaire. All 26 of the doctors performed physical examinations, blood examinations, chest radiography, and computed tomography (CT) routinely, but their frequencies varied widely among the doctors. The average frequencies of the follow-up examinations as judged from this survey are as follows: Physical and blood examinations are performed three to four times a year for the first 3 years and twice a year during the next 2 years. CT is scheduled at 6 and 12 months after resection and is repeated annually thereafter. Chest radiography is performed three to four times a year for the first 3 years and once a year thereafter, between the CT examinations.ConclusionThe follow-up programs used in clinical practice vary widely among institutions and doctors in terms of the types of examination performed and the frequencies at which they are performed.